Adhering composition and method of applying the same

ABSTRACT

A method of in-situ adhering including providing pre-gel including phenolic compound and water miscible polymers like carbohydrates, salts of anionic polysaccharide, spreading layer of pre-gel onto a surface, adding solid support, comprising cross linking agents capable of interacting with the polymer, to pre-gel, and allowing pre-gel to cure and adhere onto surface.

FIELD OF THE INVENTION

The present invention relates to adhesives. More particularly, thepresent invention relates to adhesive composition of matter and methodsof applying the same, especially in medical applications.

BACKGROUND OF THE INVENTION

Tissue adhesives have been increasingly used to enhance traditionalclosure technologies such as sutures and staples, offering improvedsealing capabilities and plugging of undesired leaks¹. However, despiterecent developments and increased clinical demand, currently availableproducts still suffer from serious drawbacks. While synthetic adhesiveshave low biocompatibility, low adherence to wet surfaces and potentialtoxicity, the biological glues are costly, often show relatively poormechanical and tissue-bonding properties, and are potentiallyimmunogenic, as most of them are based on proteins. Thus, there is agenuine unmet need for non-toxic, strong, and economical tissue sealantsto sustain internal surgical incision closure, as an adjunct to suturingor stapling. This need was the main motivation for the development ofbio-mimicking adhesives, which received increasing attention in the lastdecade.

Using an adhesive for tissue reattachments or repair procedures usuallyrequire the adhesive to be applied onto a hydrated tissue surface.Moreover, biomedical adhesives have to overcome contact withphysiological fluids such as blood or saline in order to form contactsor associations with the underlying tissue. The success of syntheticadhesives in a hydrated environment is limited, and typically requirescertain treatments and/or performing partial dehydration of the contactsurface². In contrast to synthetic materials, nature has veryeffectively conquered the limitations of sticking to wet surfaces³.Marine sessile organisms such as barnacles, reef worms, mussels, algaehave life histories that depend on their secure attachment to solidsubstrate for survival. These organisms produce and secrete adhesivesthat form permanent, strong and flexible underwater bonds to virtuallyany hard surface⁴. For example, mussels attach to wet surfaces bycreating a byssus, an extracorporeal bundle of tiny tendons that areattached distally to a foreign substratum and proximally by insertion ofthe stem root into the byssal retractor muscles. “Mussel glues” havebeen proposed to be suitable for medical applications due to their highadhesion strength and their ability to adhere to wet surfaces. However,it is clear that the commercial production of such glues is currentlynot practical, since extraction of 1 kg of the naturally existingadhesive raw materials (proteins and polypeptides) would requireprocessing five to ten million mussels¹.

An alternative and more practical method is based on taking a‘biomimetic’ approach, which entails constructing artificial materialsthat mimic natural forms. Polymeric analogs may be synthesized, as anexample, from amino acids that were identified as being functional tonaturally existing adhesive proteins. Much effort has been made tosynthesize random block copolymers, which are biomimetic approximationsof naturally existing adhesive proteins and polypeptides^(2, 8-20.)

Another effective natural adhesion mechanism exists in red and brownalgae, which produce phenolic compounds that exhibit adhesive propertiesand extraordinarily high cohesive strength. These adhesive containphenolic compounds that bind non-specifically to both hydrophobic andhydrophilic surfaces in aqueous conditions²¹. The secretion of thesephenolic compounds is coupled with peroxidase oxidation and results intheir crosslinking of cell-wall polysaccharides. Based on thoseobservations, Vreeland et al. disclosed in U.S. Pat. No. 5,520,727entitled “Aqueous algal-based phenolic type adhesives and glues” inwhich the algal-based phloroglucinol was activated and cross linked withalgal carbohydrates in order to form glue. The inventors of the presentinvention have demonstrated that formulations composed of oxidizedpolyphenol extracted from Fucus serratus, alginate and calcium ions arecapable of adhering to a variety of surfaces²³. Structural analysisusing small angle x-ray scattering (SAXS) and electron microscopy(cryo-TEM) showed that the polyphenols self-assemble into chain-likeobjects²⁴. Oxidation did not alter this overall structure, causing onlya reduction in the aggregate size. Moreover, this chain-like structuredid not change upon addition of alginate. Once calcium ions were added,a network (whose overall structure resembled that of the alginate gel)was formed.

Since the production of nature-based glues such as disclosed in Vreelandet al. and others rely on extracting natural materials from tons ofalgae, there was a need to synthetically imitate the remarkable abilityof marine algae to attach to wet solid surface in order to provideeffective adhesives having characteristics that are similar to thecharacteristics of the marine algae.

Using the biomimetic approach, the inventors of the present inventionhypothesized that the natural components of the “fucus glue” can besuccessfully replaced with commercially available analogue that providessimilar functionally. In PCT/IL2006/000289, the inventors of the presentinvention indeed showed that the monomeric unit of phloroglucinol andseveral of its derivatives to interact with polysaccharide such asalginate to form an adhesive that was shown to adhere in variouscompositions to animal tissues as well as to other surfaces.

Interactions between carbohydrates and polyphenols are not unique toalgae adhesives. Polyphenols are a large and very diverse family ofplant metabolites, characterized by the presence of more than one phenolgroup per molecule²⁵⁻³⁰.

It is needed to extend the adhesive composition beyond the biomimeticapproach and to develop adhesive composition of matter that are able toform strong interactions with surfaces, whether dry or wet, as well aswithin the network itself.

Moreover, the method of applying the adhesive materials seems to play animportant role in the ability to utilize the adhesive composition ofmatter as an effective sealant. The inventors of the present inventiondeveloped methods of applying the adhesive material that allow on-sitecuring of the adhesive and usage of bandages that form with the adhesivematerial an affective sealant especially for medical use.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide novel phenol-basedadhesives in order to further utilize them as adhesives and sealants.

It is another object of the present invention to provide various novelcompositions of matter based on phenolic compounds to be used asadhesives for bio-medical applications.

It is yet another object of the present invention to provide methods ofapplying the adhesive material on-site, where tissue repair or tissuesealing is required so as to establish effective adhesive and sealingproperties.

An additional object of the present invention is to provide acomposition of matter for a pre-gel that is applied on a solid supportto produce a sealing bandage.

It is therefore provided in accordance with a preferred embodiment ofthe present invention an adhering bandage comprising:

-   -   pre-gel that comprises        -   at least one phenol-based compound;        -   at least one water miscible polymer;    -   at least one cross linking agent capable of interacting with        said    -   at least one water miscible polymer    -   a solid support.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said at least one cross linking agent is providedwithin said solid support.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said solid support is a thin film selected from agroup of supports such as plastic, knitted mesh of fabric made fromsynthetic or natural polymer, gauze prepared form oxidized cellulose.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said pre-gel further comprises non-soluble suspendedsolids.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said suspended solids are form of particles such asfibers.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said at least one phenol-based compound can be in aform selected from monomer, non-cross linked polymer, or cross linkedpolymer.

Furthermore and in accordance with another preferred embodiment of thepresent invention, the adhering bandage further comprising at least oneactivating agent capable of promoting production of polymeric form ofsaid at least one phenol-based compound.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said at least one activating agent is selected from agroup of such as haloperoxidase (HPO) enzyme, an oxidizer, a halogensalt, and combinations thereof.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said at least one water miscible polymer is naturallyexisting form or a synthetically prepared form of carbohydrate.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said carbohydrate is selected from a group ofcarbohydrates consisting of alginic acid or salt from alginic acid.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said at least one cross linking agent are multivalentcharged materials such as polyelectrolytes, organic salts, and otherinorganic salts.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said multivalent charged materials are divalent ions.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said divalent ions are selected from a groupconsisting of divalent ions such as divalent calcium ions (Ca⁺²)supplied, for example, by calcium chloride (CaCl₂), or by a combinationof calcium carbonate (CaCO₃) and glucono-δ-lactone (GDL).

Furthermore and in accordance with another preferred embodiment of thepresent invention, said at least one phenol-based compound is selectedfrom a group consisting of phloroglucinol, Gallic acid, Morin,Naringenin, Epicatechin, Catechin, Epigallocatechin, Gallocatechin,Epigallocatechin gallate, Epicatechin gallate.

Furthermore and in accordance with another preferred embodiment of thepresent invention, the adhering bandage further comprising therapeuticmaterials such as drugs, growth factors, hormones, or any othertherapeutic agent.

It is also provided in accordance with yet another preferred embodimentof the present invention, a method of in-situ adhering comprising:

-   -   providing pre-gel that comprises a mixture of at least one        phenol-based compound and at least one water miscible polymer;    -   spreading a layer of said pre-gel onto a surface;    -   adding cross linking agent capable of interacting with said at        least one water miscible polymer to said pre-gel;    -   allowing said pre-gel to cure and adhere onto the surface;    -   providing a solid support.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said adding at least one cross linking agent isachieved by a method such as spraying, dripping, or wetting.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said at least one cross linking agent is providedwithin said solid support.

Furthermore and in accordance with another preferred embodiment of thepresent invention, the method further comprising soaking said solidsupport with said pre-gel.

Furthermore and in accordance with another preferred embodiment of thepresent invention, the method further comprising adding an acid such asslowly hydrolyzing D-gluco-δ-lactone and wherein said cross linkingagent is an insoluble salt and wherein said adding cross linking agentis achieved by blending.

Furthermore and in accordance with another preferred embodiment of thepresent invention, the method further comprising providing a solidsupport and embedding said solid support within said pre-gel.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said solid support is selected from a group of thinfilm such as plastic (e.g. PGA, poly(caprolacton) etc.), knitted mesh offabric made from synthetic or natural polymer, gauze prepared fromoxidized cellulose.

Furthermore and in accordance with another preferred embodiment of thepresent invention, the method further comprising adding an acid such asslowly hydrolyzing D-gluco-δ-lactone and wherein said cross linkingagent is an insoluble salt and wherein said adding cross linking agentis achieved by blending.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said solid support is coated with said cross linkingagent.

Furthermore and in accordance with another preferred embodiment of thepresent invention, the method further comprising drying said pre-gel.

Furthermore and in accordance with another preferred embodiment of thepresent invention, the method further comprising drying said pre-gelwith said cross linking agent.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said adding at least one cross linking agent isproviding the cross linking agent to said solid support and furthercomprising releasing said cross linking agent from said solid support.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said at least one phenol-based compound can be in aform selected from monomer, non-cross linked polymer, or cross linkedpolymer.

Furthermore and in accordance with another preferred embodiment of thepresent invention, the method further comprising at least one activatingagent capable of promoting production of polymeric form of said at leastone phenol-based compound.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said at least one activating agent is selected from agroup of such as haloperoxidase (HPO) enzyme, an oxidizer, a halogensalt, and combinations thereof.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said at least one water miscible polymer is naturallyexisting form or a synthetically prepared form of carbohydrate.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said carbohydrate is selected from a group ofcarbohydrates consisting of alginic acid or salt from alginic acid.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said at least one cross linking agent are multivalentcharged materials such as polyelectrolytes, organic salts, and otherinorganic salts.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said multivalent charged materials are divalent ions.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said divalent ions are selected from a groupconsisting of divalent ions such as divalent calcium ions (Ca⁺²)supplied, for example, by calcium chloride (CaCl₂), or by a combinationof calcium carbonate (CaCO₃) and glucono-δ-lactone (GDL).

Furthermore and in accordance with another preferred embodiment of thepresent invention, said at least one phenol-based compound is selectedfrom a group consisting of Phloroglucinol, Gallic acid, Morin,Naringenin, Epicatechin, Catechin, Epigallocatechin, Gallocatechin,Epigallocatechin gallate, Epicatechin gallate.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said surface is a surface selected from a group oftissue, graft, or organ.

There is also provided in accordance with yet another preferredembodiment of the present invention, a use of the adhering bandagewherein said adhering bandage is usable under dry or wet conditions.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said adhering bandage is usable under dry conditions,for adhering a first surface to a second surface, wherein each of saidfirst surface and said second surface is dry.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said adhering bandage is usable under wet conditions,for adhering a first surface to a second surface, wherein at least oneof said first surface and said second surface is wet.

Furthermore and in accordance with another preferred embodiment of thepresent invention, at least one of said first surface and said secondsurface is a body part or a component thereof, of a human or animalsubject.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said component is a tissue.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said adhering bandage is usable as a sealant orsealing agent, for sealing or closing an opening in asurface.\Furthermore and in accordance with another preferred embodimentof the present invention, said sealing or closing takes place under dryor wet conditions.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said surface having said opening is a body part or acomponent thereof, of a human or animal subject.

Furthermore and in accordance with another preferred embodiment of thepresent invention, said component is a tissue.

BRIEF DESCRIPTION OF THE FIGURES

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of the preferred embodiments of the present invention only,and are presented in the cause of providing what is believed to be themost useful and readily understood description of the principles andconceptual aspects of the invention. In this regard, no attempt is madeto show structural details of the invention in more detail than isnecessary for a fundamental understanding of the invention, thedescription taken with the drawings making apparent to those skilled inthe art how the several forms of the invention may be embodied inpractice.

In the drawings:

FIG. 1 illustrates several chemical structures of selected phenols thatcan be utilized as adhesive base materials, in their monomeric orpolymeric forms.

FIG. 2 illustrates tensile strength required to separate two porcinetissue strips adhered by polyphonel-based adhering materials inaccordance with preferred embodiments of the present invention, Theright bar represents the tensile strength obtained with Tisseel®, acommercial fibrin sealant.

FIG. 3 illustrates tensile strength of KI-oxidized adhesive (dark grey)in accordance with a preferred embodiment of the present inventioncompared to KI-oxidized Fucus glue (light grey) to various substrates.

FIG. 4 Illustrates tensile strength of KI-oxidized adhesive (light grey)and non oxidized adhesive (dark grey) in accordance with preferredembodiments of the present invention to various substrates.

FIG. 5 illustrates SAXS curves from alginate and calcium ( ) andphloroglucinol-containing adhesive material (⋄). The solid linesrepresent the fit results of the “broken rod linked by a flexible chain”model. Best-fit parameters are: Alginate gel: ξ=25 Å, R₁=8.2 Å, R₂=118Å, C=8.1. Biomimetic glue: ξ=22 Å, R₁=7.8 Å, R₂=85 Å, C=6.7.

FIG. 6 illustrates ITC data for (A) Phloroglucinol (8 mg/ml, 63.4 mM)titrated into LF-200S alginate (5 mg/ml, 0.0236 mM). Lower plot shows afit to a two-site model. (B) Epicatechin (2 mg/ml, 68.8 mM) titratedinto LF-200S alginate (5 mg/ml, 0.0236 mM). Lower plot shows a fit to aone-site model.

DETAILED DESCRIPTION OF THE INVENTION AND THE FIGURES

The present invention provides novel composition-of-matter of adhesivesand methods of applying thereof in a wide variety of different fields,and in particular, in the health care fields of medicine, dentistry, andveterinary science. The present invention is especially applicable foruse by health care providers, such as medical, dental, and veterinary,surgeons, in procedures for reattaching or repairing body parts orcomponents thereof, such as tissues of human or animal subjects,especially under wet conditions, but not necessarily. Thecomposition-of-matter of the present invention, applied as an adhesive,may also function, and be usable, as a sealant or sealing agent, forsealing or closing an opening in a surface, for example, for preventingflow of liquid or/and gaseous fluid. Such a sealant or sealing agent canbe used in a wide variety of applications, for example, for sealing orclosing an opening in a medical device, of an aquarium, or of a widevariety of other objects or entities.

A main aspect of the present invention is providing a composition ofmatter that comprises at least one cross-linked form of a water misciblepolymer and at least one phenol-based material. The phenol-basedmaterial can be in its monomeric or polymeric form.

Accordingly and optionally, at least one activating agent is used in thecomposition for promoting reaction and possible cross-linking, or/andoxidation, or/and some other modification, of any of the phenol typecompound so as to produce its polymeric form. Such activating agentsare, for example, haloperoxidase (HPO) enzyme, an oxidizer, a halogensalt, and combinations thereof.

The water miscible polymer is essentially any type or kind of naturallyexisting polymer or synthetically prepared polymer which is miscible inwater. Exemplary water miscible polymers can be naturally existing, orsynthetically prepared, form of a carbohydrate (polysaccharide), such asalginic acid, or/and alginic acid itself. More preferably, the watermiscible polymer is a naturally existing, or synthetically prepared,salt form of a carbohydrate (polysaccharide), such as a salt form ofalginic acid, being an alginate. The water miscible polymer is in across-linked form. For an embodiment of the composition-of-matter of thepresent invention, wherein the water miscible polymer is an alginate, oralginic acid, preferably, the alginate, or the alginic acid, iscross-linked via interaction with divalent ions, for example, divalentcalcium ions (Ca⁺²) supplied, for example, by calcium chloride (CaCl₂),or by a combination of calcium carbonate (CaCO₃) and glucono-δ-lactone(GDL).

Another main aspect of the present invention is providing a pre-gel thatmay be usable as an adhesive in combination with a solid support. Thecombined pre-gel and solid support may be functional and usable as asealant or sealing agent, for sealing or closing an opening in a surfaceso as to prevent as an example flow of a fluid through the sealed orclosed portion of the surface. The sealing or closing may take placeunder dry or wet conditions.

Reference is made to FIG. 1 illustrating several chemical structures ofselected phenols that can be utilized as adhesive base materials. Thestructures are shown in their monomeric form, however, their polymericform is being used also. These examples are of non-toxic polyphenols canbe utilized as base material for an adhesive that can be used in themedical field. The exemplary polyphenols shown in FIG. 1 are appropriateanalogues that can replace the natural components of glues such as“fucus glue” or others that were mentioned in the background section ofthis document. Most importantly, such commercially available polyphenolsare purchased from Sigma as well as other manufacturers. A largeselection of polyphenols was exploited in order to evaluate theinfluence of their molecular parameters (molecular weight, abundance ofphenolic groups within the structure, molecular flexibility) on theinteractions with the carbohydrate on one hand, and on the propertiesand the performance of the adhesive material, on the other.

It should be noted that some polyphenols are only sparingly soluble inwater; however preliminary experience has shown that occasionally thesolubility in polysaccharide solutions is higher compared to water.

One example for a polysaccharide that is included in the preparation ofthe adhesive material of the present invention is a commercial alginatesupplied by FMC Biopolymer. Alginate is structured as a block copolymerwith blocks of α-L-gluronic acid (G) and β-D-mannuronic acid (M)alternating interrupted by regions of more random distribution of M andG units. Sizes of the three blocks can vary over a wide range, givingrise to alginates of different properties. Two types of sodium alginate,Protanal LF 200 S with G content of ˜70% and Protanal HF 120 RBS with Gcontent of ˜50% were used. It should be noted again that this alginatewas used as an exemplary alginate and any other miscible polymer can beused in order to prepare the adhesive material.

Alternatively and optionally, alginate can be replaced with apolysaccharide that can undergo gelation upon interaction with ions andchanges in temperature. In this case, the gels are dense cross-linkedpolymeric matrices that can hinder solute diffusion. Therefore drugs,growth factors, hormones, or any other therapeutic agents, can beentrapped within the cured glue or sealant and will be slowly releasedin the site of application.

In the specific case of alginate, gelation is induced by the addition ofmultivalent ions. For example, calcium ions were added either as CaCl₂,or by blending the mixture with insoluble salt such as CaCO₃ or CaEGTAfollowed by addition of the slowly hydrolyzing D-gluco-δ-lactone (GDL).Salts, which were previously shown to alter alginate-polyphenolcomplexation³¹, were added to some of the formulations. Optionally,other sources of multivalent ions are polyelectrolytes, organic salts,and other inorganic salts (e.g. Al, Ba).

As mentioned herein before, the method of applying the adhesive materialis one of the main aspects of the present invention and special focus isgiven to combining solid support to the adhesive material so as to forman adhering bandage. In the following text, the term “pre-gel glue”refers to a composition of matter that typically contains apolysaccharide such as alginate, polyphenol, salt and water, at anygiven ratios. The pre-gel may also contain multivalent ions, yet it hasrelatively low viscosity since the polysaccharide is not fully gelled.The solid support may be a thin film made from plastic (e.g. PGA,poly(caprolacton) etc.), knitted mesh of fabric made from synthetic ornatural polymer, gauze prepared form oxidized cellulose etc. Any othercomposition of the pre-gel as claimed in the present invention can beused without limiting the scope of the present invention.

Optionally, the pre-gel glue may contain non-soluble suspended solids inthe form of particles, fibers etc. which are added to enhance themechanical strength of the glue.

The pre-gel glue is cured (solidified) so as to form an adhesivematerial typically due to the addition of multivalent ions or anothermethod which induces polysaccharide gelation.

Several methods are used to apply the glue on site that requires tissuerepair or tissue sealing as follows:

(1) Spreading a layer of the pre-gel on the tissue or surface followedby hardening (curing) of the polysaccharide which can be achieved, forexample, by spraying, dripping, or wetting the pre-gel with an aqueoussolution containing multivalent ions.(2) Blending the pre-gel solution with insoluble salt of multivalentions (e.g. CaCO₃ or CaEGTA) and an acid (e.g. the slowly hydrolyzingD-gluco-δ-lactone (GDL)), then spreading a layer on the tissue or thesurface. The pre-gel hardens with time due to the slow dissolution ofthe multivalent salt.(3) Providing a patch for solid support such as a thin film made fromplastic (e.g. PGA, poly(caprolacton) etc.), knitted mesh of fabric madefrom synthetic or natural polymer, gauze prepared from oxidizedcellulose etc. The patch or solid support is soaked with a pre-gel andplaced on the tissue. Alternatively, a layer of pre-gel is spread on thetissue or the surface and the solid support is embedded within it.Finally, the polysaccharide is hardened by spraying, dripping, orwetting the pre-gel with an aqueous solution containing multivalentions.(4) Blending the pre-gel solution with insoluble salt of multivalentions (e.g. CaCO₃ or CaEGTA) and an acid (e.g. slowly hydrolyzingD-gluco-d-lactone (GDL) or acetic acid). A patch or solid support suchas a thin film made from plastic (e.g. PGA, poly(caprolacton) etc.),knitted mesh of fabric made from synthetic or natural polymer, gauzeprepared from oxidized cellulose etc. is soaked with a the pre-gel andplaced on the tissue or the surface.Alternatively, a layer of pre-gel is spread on the tissue and the solidsupport is embedded within it. The pre-gel hardens with time due to theslow dissolution of the multivalent salt.(5) Coating a solid support such as a thin film made from plastic (e.g.PGA, poly(caprolacton) etc.), knitted mesh of fabric made from syntheticor natural polymer, gauze prepared from oxidized cellulose as an examplewith multivalent ions. A layer of pre-gel is spread on the tissue or thesurface and the solid support is embedded in it. The pre-gel hardenswith time due to the slow release of the multivalent salt from the solidsupport.(6) Preparing a dry film made from the components of the pre-gel.Multivalent ions are used to coat a solid support as described above,and the solid film is attached on top of the dried pre-gel. Both filmsare placed on a hydrated tissue. The hydration of the films leads totheir adhering to the tissue. At the same time, the pre-gel hardens withtime due to the slow release of the multivalent salt from the solidsupport.(7) Preparing a dry film made from the components of the pre-gel, withadded multivalent ions in a dry form. The dry multivalent ions may beincorporated in the pre-gel formulation or as a different layer of thedry film (i.e. multilayer film). The film is placed on a hydrated tissueor surface. The hydration of the film leads to its adhering to thetissue. At the same time, the pre-gel hardens with time due to the slowdissolution release of the multivalent salt.

Regardless of the method by which the bandage was prepared, additionaltop layers (typically made of polymers) may be added to the finishedbandage to provide extra mechanical support, provide an inert layer thatwill separate between the inner bandage layers and the surroundingenvironment etc. The additional layers, by no means, limit the scope ofthe present invention.

In order to show the performance of the composition of matter of thepresent invention as adhesives, several adhesive properties were testedfor several of the polyphenol-based adhesive materials and the adhesivebandage.

Experimental Methods Adhesive Properties

Adhesive strength is characterized using shear lap and tensile tests.The samples holders and specimens preparation was described in theinventor's PCT publication WO 06/092798. Briefly, two identical sampleholders are used to prepare a specimen. The adhesive material is fixedto both sample holders using synthetic glue. Next, a measured volume ofthe studied composition of matter is applied onto one adherent,immediately covered with the other, clamped, and cured for a specifiedtime period. Adhesion tests are performed using a Lloyd tensile machineequipped with a 50N load cell. 10 specimens are prepared for eachformulation and a statistical analysis is performed. From practicalreasons, and following previous works, specimen preparation and adhesionassays are performed at room temperature with no attempt to simulategluing under water.^(2, 8). However, evaluation of the influence ofcuring in humid environment was established on the final results.

The adhesion to different well-defined clean test substrates (e.g.glass, metals, and different types of plastic) is characterized for thedifferent composition of matters.

Additional adhesion assays were performed using tissues obtained from alocal slaughterhouse. These provide better sense for the glue's abilityto adhere to moist and flexible surfaces.

Rheological Measurements

Rheological measurements were conducted using a Rheometric ScientificARES strain-controlled rheometer fitted with a 50-mm cone-and-platefixture and equipped with anti-evaporator cover that prevents sampledehydration. Oscillatory shear experiments were performed within thelinear viscoelastic regime, where the dynamic storage modulus (G′) andloss modulus (G″) are independent of the strain amplitude. Calculationsare done using the RSI Orchestrator 6.5.1 software. The critical calciumconcentration required to induced a sol-gel transition at a givencomposition of matter concentration are determined from the appearanceof a power law of the dynamic moduli³⁵, as proposed by Winter andChambon^(36, 37). Moduli exhibiting power law dependency on thefrequency ω of G′ ˜ω^(1.5) and G″ ˜ω¹ are characteristic to aviscoelastic fluid according to the Rouse-Zimm theory. Above the gelpoint, G′ becomes larger than G″ with a plateau appearing in the G′ vs.ω curve at the low frequency range.

Wetting Properties

Advancing contact angles of the liquids on the test surfaces aremeasured by a computerized contact angle analyzer (CAM200, KSV,Helsinki, Finland). A micro Hamilton syringe were used to create thedrop on the surface and to continuously add liquid during themeasurement. The surface tension of the aqueous solutions was measuredby the ring method. The work of the adhesion W_(adh) is calculatedaccording to the Young-Dupré equation W_(adh)=γ_(LV)(1+θ)⁸. The wettingproperties of both polyphenol solution and polyphenol/alginate wasstudied.

Miscible Polymer/Polyphenol Interactions

¹H NMR Spectroscopy: Comparing spectra obtained from the composition ofmatter solutions with spectra obtained from the individual componentsprovide a qualitative indication to intramolecular interactions²⁴.

Equilibrium Dialysis was previously used to determine the extent ofbinding of polyphenols to macromolecules²⁶. Binding assays includedialyzing 0.5 ml polyphenol solution, contained in a Spectra/Por®dialysis tubing (Spectrum Laboratories®, CA, USA) against stirredpolysaccharide solution³⁸. Once equilibrium is achieved, the polyphenolconcentration in both cells is determined spectrophotometrically. Thepartition coefficient is calculated from a mass balance. A controlexperiment (dialysis against water) is used to ensure that polyphenol isnot adsorbed by the cellulose membrane installed in the dialysis tube.Isothermal Titration Calorimetry (ITC) is used to quantify the magnitudeof the interactions between the polyphenols and the polysaccharides(alginate). ITC is a technique that allows studying the heat ofinteraction between two molecules. Often, a “ligand” is titrated to asolution of a “macromolecule”, and the heat released upon theirinteraction, Q, is monitored over time. As the two elements interact,heat is released or absorbed in direct proportion to the amount ofassociation/dissociation that occurs. When the ligand in the cellbecomes saturated with added macromolecule, the heat signal diminishesuntil only the background heat of dilution is observed. ITC measurementswere performed using a VP-ITC Microcalorimeter (MicroCal Inc.). ITCexperiments include injecting known amounts of polyphenol solution intoan aqueous alginate solution. Appropriate reference experiments(injection of buffer into alginate and polyphenol dilution) areperformed as well. The experimental data were analyzed using thesoftware provided with the instrument, which calculates the bindingconstant from the slope of the heat vs. injected amount at thesaturation point. In a case of small binding energies, one of theanalysis scheme previously applied by the inventors of the presentinvention and others³⁹⁻⁴¹ was followed. Data obtained from ITC andequilibrium binding experiments can be further used to calculate thefree energy and entropy of binding^(26, 40). This approach providesadditional insight into the binding mechanism and in particularhighlights the related importance of the entropy driven hydrophobicinteractions and the enthalpy driven hydrogen bonding.

Nanostructure Characterization

Nanostructure characterization allows assessment of the estimated modelof polyphenols and alginate that is hypnotized to form a nanocompositematerial. Moreover, the structural parameters of such nanocompositemight have a significant impact on its properties.SAXS measurements are mostly performed using a slit-collimated compactKratky camera (A. Paar Co.) equipped with a linear position sensitivedetector system (Raytech), with pulse-height discrimination and amultichannel analyzer (Nucleus). A total of 3000 or more counts for eachchannel are collected in order to obtain a high signal to noise ratio.Primary beam intensities is determined using the moving slit method⁴²and subsequently using a thin quartz monitor as a secondary standard.The scattering curves, as a function of the scattering vector q=4π sinθ/λ (where 2θ and λ are the scattering angle and the wavelength,respectively), are corrected for counting time and for sample absorptionand the background is subtracted. Desmearing procedure is performedaccording to the Indirect Transformation Method⁴³ using the ITP program.Data analysis includes fitting the desmeared curve to an appropriatemodel using a least-squares procedure. As a starting point, theinventors fitted the model used for the algal-born glue²⁴, known as“broken rod linked by a flexible chain” model^(44, 45):

${I(q)} \propto {{S(q)}{P(q)}} \propto {\underset{\underset{S{(q)}}{}}{\frac{1}{1 + {C\; {\exp \left( {{- \xi^{2}}q^{2}} \right)}}}}\underset{\underset{P{(q)}}{}}{\frac{1}{q^{2}}{\sum\limits_{i}{k_{i}{q\left\lbrack \frac{J_{1}\left( {q\; R_{i}} \right)}{q\; R_{i}} \right\rbrack}^{2}}}}}$

Where P(q) is the form factor of cylindrical elements specified bycross-sectional radii R_(i) and relative weights of K_(i). Theelectrostatic interactions are taken into account by the structurefactor S(q) that assumes a Gaussian-type interaction potential specifiedby the correlation length ξC is a an adjustable parameter representingthe strength of interaction, which depends on the second virialcoefficient and the polymer concentration.^(44 45.)Cryo-Transmission Electron Microscopy (cryo-TEM) is used in parallel fordirect visualization of the nanostructure. Ultra-fast cooled vitrifiedspecimens, prepared at controlled conditions of 25° C. and 100% relativehumidity as described elsewhere⁴⁶, are studied in a Philips CM120cryo-TEM operating at 120 kV. An Oxford CT3500 cooling-holder systemthat keeps the specimens at about −180° C. was used. Low electron-doseimaging is performed with a Gatan Multiscan 791 CCD camera, using theGatan Digital Micrograph 3.1 software package.

Pulsatile Flow System.

A flow system was built in order to serve as a model system thatimitates the forces working on blood vessels in the human body, thusproviding a more realistic method of testing the adhesives. The flowsystem creates pressure swing between the systolic and diastolic bloodpressures. The inventors tested punctured aorta originated from bovineor swine as a substrate for testing.

In order to analyze the results, an experimental parameter named sealingratio (SR) was defined as

${S\; R} = {1 - {\frac{{Flow}\mspace{14mu} {through}\mspace{14mu} {sealed}\mspace{14mu} {hole}}{{Flow}\mspace{14mu} {rate}\mspace{14mu} {through}\mspace{14mu} {unsealed}\mspace{14mu} {hole}}.}}$

T-Peel Test

T-peel test is based on ASTM F-2256-05 that was developed as testingmethod for surgical adhesives and sealants.The samples used for the T-peel test are of constant dimensions.The solid support dimensions are 150

mm length and 25

mm width. The dimensions of the sample tissue, which was an artery,equal to those of the solid support.1.75 ml of adhesive was applied on the tissue. The hardening film isthen applied and a weight of 1 kg is placed over it for one minute. Thesample is then placed in the extension test machine and the test isconducted.The results are analyzed as described in ASTM F-2256-05.

Experimental Results

Reference is now made to FIG. 2 illustrating tensile strength requiredto separate two porcine tissue strips adhered by polyphonel-basedadhering materials in accordance with preferred embodiments of thepresent invention. The right bar represents the tensile strengthobtained with Tisseel®, a commercial fibrin sealant, for comparison.Results show firm adhesion between the tissue surfaces after adherenceof the tissues with a phenolic adhering material; wherein the tissuesare porcine muscle tissues.

Different types of polyphenols were used to demonstrate the feasibilityof the present invention. The composition of the differentpolyphenol-based adhesive materials shows significant adhesionproperties. It should be noted that some of the polyphenol-basedadhesive materials showed better adhesion to the tissue than Tisseel®, acommercial fibrin sealant.

Reference is now made to FIG. 3 illustrating tensile strength ofKI-oxidized adhesive (dark grey) in accordance with a preferredembodiment of the present invention compared to KI-oxidized Fucus glue(light grey) to various substrates. Tensile strength of alga-born glue,composed of components extracted from Fucus Serratus (15 mg/ml Alginate,5 mg/ml polyphenol, 4 mM ca ions) are compared to these of a biomimeticcomposition in matter in which the algae polyphenol was replaced withphloroglucinol and the algae alginate with Protanal LF 200 S alginate.All samples were oxidized by adding 0.75 U/ml BPO, 0.44% H₂O₂ and 4.4mg/ml KI. Oxidation was verified using NMR (data not shown). Sampleswere cured for 20 minutes. The adhesive strengths of the biomimeticadhesive was comparable to that of the alga-born one. Moreover, bothglues seem to adhere better to the more hydrophobic surfaces.

A preliminary examination of the effect of oxidation was achieved byrepeating the experiment described in (A) while omitting the oxidationagents. Oxidized formula adhered better to the Teflon while thenon-oxidized formula adhered better to the glass and Mylar (FIG. 4).

Reference is now made to FIG. 5 illustrating SAXS curves from alginate(15 mg/ml) and calcium (4 mM) polyphenol, 0.75 U/ml BPO, 0.44% H₂O₂, 4.4mg/ml KI, 15 mg/ml alginate and 4 mM calcium ions was ( ) andphloroglucinol-containing adhesive material (alginate 15 mg/ml,phlologlucinol 5 mg/ml, Ca 4 mM) (⋄). The solid lines represent the fitresults of the “broken rod linked by a flexible chain” model. Best-fitparameters are: Alginate gel: ξ=25 Å, R₁=8.2 Å, R₂=118 Å, C=8.1.Biomimetic glue: ξ=22 Å, R₁=7.8 Å, R₂=85 Å, C=6.7. The SAXS resultscomparison demonstrates the existence of alginate-phloroglucinolinteractions in a biomimetic composition of matter. The overallstructure of the glue resembles that of alginate gel. Yet, since thescattering from phloroglucinol by itself was very weak and a scatteringcurve could not be obtained, the differences between the two curves canbe attributed to interactions between the alginate and thephloroglucinol. As in the case of algal-born glue²⁴, both curves werewell fitted by the “broken rod linked by a flexible chain” model. Theanalysis of the SAXS data showed that the two samples differ by thecorrelation length ξ, the adjustable parameter C and R₂ that representsthe alginates' tendency to form large aggregates⁴⁷. Smaller correlationlength and adjustable parameter were obtained for the biomimetic glue,thus indicating weaker electrostatic repulsion and lower hydrophilicitycompared to alginate gel⁴⁸.

Reference is now made to FIG. 6 illustrating ITC data for (A)Phloroglucinol (8 mg/ml, 63.4 mM) titrated into LF-200S alginate (5mg/ml, 0.0236 mM). Lower plot shows a fit to a two-site model. (B)Epicatechin (2 mg/ml, 68.8 mM) titrated into LF-200S alginate (5 mg/ml,0.0236 mM). Lower plot shows a fit to a one-site model. Two preliminaryITC measurements detected heat changes during injection of polyphenolinto alginate solution, thus providing additional evidence tointeractions between the two materials that comprises the adhesivematerial. Moreover, the affect of the polyphenol's molecular structureis evident from a comparison between the experiments.

As mentioned herein above, many different configurations of dry film maybe used in order to apply the adhering material of the present inventionwith their formulations. The composition of matter of the adheringmaterial can differ in their physical form, composition and preparationmethod. The following examples include two different preparation methodsand two different compositions.

Air drier film, a solution containing 35 mg/ml alginate (LF200S), 10mg/ml PHG and 1 mg/ml colorant was cast into molds with different depthsand air dried for 24 hrs. The film was peeled and use in conjunctionwith ORC containing ˜1 mg/cm² CaCl2 as hardener. The sealing ratio wasmeasured using the flow system mentioned above. A sealing ratio close to1 at 120 mmHg was achieved for 14 samples.

Freeze dried film are prepared from a solution containing alginate, PHGand colorant that were cast into molds with different widths. Thesolutions were freeze by liquid nitrogen, however, can be freeze also bysolid carbon dioxide or any other cooling method. In some cases, asecond layer was applied, wherein this layer contained PVA/PEG/CACl₂mixture. The samples were than inserted into freeze drier untilcompletely dried.

Single layer samples—these patches were used in conjunction withexternal ORC hardening system containing ˜1 mg/cm2 CaCl₂. The solutionused for casting these films contained 35 mg/ml alginate (LF200S), 10mg/ml PHG and 0.3 mg/ml colorant (acid green 25 or other such as indigocarmine)

The sealing ratio for 1.5 mm thick film was approximately 1 at 200 mmHg,The number of repetitions was 22.

Multilayer samples—a first 1.5 mm thick layer was cast using solutioncontaining 35 mg/ml alginate (LF200S), 10 mg/ml PHG and 0.3 mg/mlcolorant (acid green 25 or other such as indigo carmine). After thefirst layer was frozen, a second layer was cast using a solutioncontaining PVA/PEG/CaCl₂ in the following concentrations, 34.4/15.6/20mg/ml. The second layer was allowed to freeze and the multilayer filmwas placed on freeze drier until completely dried. The sealing ratio forthis composition was found to be approx. 1 at 200 mmHg.

Although most of the results shown herein deal with alginate as apreferred water miscible polymer, a carbohydrate, other water misciblepolymers were tested. As an example, a T-peel test was conducted forpolygalacturonic acid as another preferred carbohydrate that showsT-peel strength of 0.076N/cm.

For any of the above described preferred embodiments or formulations ofthe composition of matter of the present invention generally usable asan adhesive; the adhesive may be functional and usable as sealant orsealing agent, for sealing or closing an opening of a dry or wetsurfaces. For example, for preventing flow of a liquid or/and gaseousfluid through the sealed or closed portion of the surface. Accordingly,the sealing or closing may take place under dry or wet conditions. Thesurface having the opening which is sealed or closed may be a body partor a component thereof (e.g. a tissue), of a human or animal subject.Such a sealant or sealing agent can be used in a wide variety ofapplications, for example, for sealing or closing an opening in a dry orwet body part, or in a dry or wet surface of a medical device, of anaquarium, or of a wide variety of other objects or entities.

Any of the above described preferred embodiments or formulations of thecomposition of matter of the present invention are generally usable asan adhesive, of a variety of different types of surfaces, under dry orwet conditions.

In particular, the composition of matter of the present invention isgenerally usable as an adhesive under dry conditions, for example, foradhering a first surface to a second surface, wherein both surfaces aredry. Alternatively and advantageously, the composition of matter of thepresent invention is generally usable as an adhesive under wetconditions, for example, for adhering a first surface to a secondsurface wherein the first surface is wet and/or the second surface iswet.

Any of the above described preferred embodiments or formulations of thecomposition of matter of the present invention is generally usable as asealant or an adhesive in any condition, wet or dry, and on any kind ofsurface, in a wide variety of different fields such as health care,medicine, dentistry, veterinary, as well as other general fields such ascommercial, laboratory or home use.

It should be clear that the description of the configurations andattached Figures set forth in this specification serves only for abetter understanding of the invention, without limiting its scope ascovered by the following Claims.

It should also be clear that after reading the present specification askilled person, can make adjustments or amendments to the attachedFigures and above described configurations that would still be coveredby the following Claims.

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1.-46. (canceled)
 47. A composition comprising: a) pre-gel thatcomprises at least one phenol-based compound, and at least one watermiscible polymer selected from at least one of a naturally existing formof a carbohydrate, a synthetically prepared form of carbohydrate and asalt of an anionic polysaccharide; b) a solid support, comprising atleast one cross linking agent capable of interacting with said at leastone water miscible polymer, wherein the pre-gel applied to a surface isconfigured to harden and adhere to the surface upon contact of the solidsupport with the pre-gel.
 48. A composition comprising: a) pre-gel thatcomprises at least one phenol-based compound, at least one watermiscible polymer selected from at least one of a naturally existing formof a carbohydrate, a synthetically prepared form of carbohydrate and asalt of an anionic polysaccharide, and a cross linking agent comprisingat least one water-insoluble salt of multivalent ions capable ofinteracting with said at least one water miscible polymer, b) a solidsupport, and c) at least one trigger compound capable of triggeringrelease of multivalent ions from the cross linking agent into thepre-gel, wherein upon blending the pre-gel with the trigger compound thepre-gel hardens and forms an adhesive material.
 49. The composition asclaimed in claim 47, wherein said solid support comprises a thin filmselected from one or more of a group of supports comprising plastic,knitted mesh of fabric made from synthetic or natural polymer, and gauzeprepared from oxidized cellulose.
 50. The composition as claimed inclaim 48, wherein said solid support comprises a thin film selected fromone or more of a group of supports comprising plastic, knitted mesh offabric made from synthetic or natural polymer, and gauze prepared fromoxidized cellulose.
 51. The composition as claimed in claim 47, whereinsaid pre-gel further comprises non-soluble suspended solids.
 52. Thecomposition as claimed in claim 48, wherein said pre-gel furthercomprises non-soluble suspended solids.
 53. The composition as claimedin claim 51, wherein said suspended solids are form of particlescomprising fibers.
 54. The composition as claimed in claim 62, whereinsaid suspended solids are form of particles comprising fibers.
 55. Thecomposition as claimed in claim 47, wherein said at least one crosslinking agent comprise multivalent charged materials selected from oneor more of a group comprising polyelectrolytes, organic salts, andinorganic salts.
 56. The composition as claimed in claim 55, whereinsaid multivalent charged materials are salts of divalent ions.
 57. Thecomposition as claimed in claim 47, further comprising therapeuticmaterials selected from one or more of a group comprising drugs, growthfactors, and hormones.
 58. The composition as claimed in claim 48,further comprising therapeutic materials selected from one or more of agroup comprising drugs, growth factors, and hormones.
 59. Thecomposition of claim 48, wherein the trigger compound is selected from agroup comprising D-gluco-d-lactone (GDL) salts and acetic acid,
 60. Amethod of in-situ adhering comprising: providing pre-gel that comprisesa mixture of at least one phenol-based compound and at least one watermiscible polymer selected from at least one of a naturally existing formof a carbohydrate, a synthetically prepared form of carbohydrate and asalt of an anionic polysaccharide; spreading a layer of said pre-gelonto a first surface; adding a solid support, comprising at least onecross linking agent capable of interacting with said at least one watermiscible polymer, to said pre-gel; allowing said pre-gel to cure andadhere onto the first surface,
 61. A method of in-situ adheringcomprising: providing pre-gel that comprises at least one phenol-basedcompound, at least one water miscible polymer selected from at least oneof a naturally existing form of a carbohydrate, a synthetically preparedform of carbohydrate and a salt of an anionic polysaccharide, and across linking agent comprising at least one water-insoluble salt ofmultivalent ions capable of interacting with said at least one watermiscible polymer; spreading a layer of said pre-gel onto a firstsurface; adding a solid support to said pre-gel; adding to and blendingwith the pre-gel at least one trigger compound capable of triggeringrelease of multivalent ions from the cross linking agent into thepre-gel, allowing the pre-gel to cure and adhere to the first surface.62. The method as claimed in claim 60, wherein said curing and adheringis achieved by a method selected from one or more of a group comprisingspraying, dripping, and wetting the solid support with said crosslinking agent.
 63. The method as claimed in claim 60, wherein said atleast one cross linking agent is provided within said solid support. 64.The method as claimed in claim 60, further comprising soaking said solidsupport with said pre-gel.
 65. The method as claimed in claim 60,further comprising embedding said solid support within said pre-gel. 66.The method as claimed in claim 60, wherein said solid support is coatedwith said cross linking agent.
 67. The method as claimed in claim 60,further comprising drying said pre-gel.
 68. The method as claimed inclaim 14, wherein said first surface is a surface selected from a groupof tissue surface, graft surface, and organ surface.
 69. The method asclaimed in claim 61, wherein said first surface is a surface selectedfrom a group of tissue surface, graft surface, and organ surface. 70.The method of adhering as claimed in claim 60, wherein said adhering isunder dry or wet conditions.
 71. The method of claim 70, furthercomprising adhering the first surface to a second surface, wherein eachof said first surface and said second surface is dry.
 72. The method ofclaim 70, further comprising adhering the first surface to a secondsurface, wherein at least one of said first surface and said secondsurface is wet.
 73. The method of claim 72, wherein at least one of saidfirst surface and said second surface is a body part or a componentthereof, of a human or animal subject.
 74. The method of claim 73,wherein said component is a tissue.
 75. The method of claim 60, whereinsaid adhering comprises sealing or closing an opening in the firstsurface.
 76. The method of claim 75, wherein said sealing or closingtakes place under dry or wet conditions.
 77. The method of claim 75,wherein said surface having said opening is a body part or a componentthereof, of a human or animal subject.
 78. The method of claim 77,wherein said component is a tissue.
 79. A method of treating an subjectcomprising: spreading a pre-gel onto a tissue of the subject, thepre-gel comprising a mixture of at least one phenol-based compound andat least one water miscible polymer selected from at least one of anaturally existing form of a carbohydrate, a synthetically prepared formof carbohydrate and a salt of an anionic polysaccharide; adding a solidsupport, comprising at least one cross linking agent capable ofinteracting with said at least one water miscible polymer, to saidpre-gel; allowing said pre-gel to cure and adhere onto the tissue.